Summary

Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determinant controlling the functional integrity of ESCs and EpiSCs.

The ESC state corresponds to the naïve ground state of the preimplantation epiblast, whereas the EpiSC state corresponds to the primed state of the postimplantation epiblast (Hanna et al., 2010; Niwa, 2007; Lanner and Rossant, 2010; Nichols and Smith, 2009), thus implying that ESCs should be converted to a primed epiblast-like state before definitive differentiation into germ layers occurs. However, despite significant advancements, the regulatory mechanisms that control the state and differentiation capability of ESCs and EpiSCs remain to be fully clarified. Here, we have studied whether the transcription factor Otx2, which is required at multiple steps in brain development and neuronal differentiation (Simeone et al., 1992; Simeone et al., 2002; Simeone et al., 2011), might be functionally relevant in ESCs and EpiSCs, where it is expressed. We found that Otx2 is crucially required to maintain the ESC metastable state by opposing self-renewal and predisposing the cells to differentiation. Moreover, Otx2 stabilizes the transition from naïve ESCs to primed EpiSCs in cooperation with BMP4 and Fgf2 and is required for telencephalic and mesencephalic differentiation of ESC-derived neural progenitors.

MATERIALS AND METHODS

Generation of ESC lines

The ESC lines were generated in E14Tg2a cells. In the Otx2–/– ESC line, the coding exons of Otx2 alleles have been replaced with lacZ and GFP (Acampora et al., 1995; Acampora et al., 2009) (supplementary material Fig. S1A-C). In the R26Otx2/Otx2 ESC line, a cassette including full-length Otx2 coding cDNA followed by an IRES GFP sequence is inserted into the Rosa26 locus (Di Salvio et al., 2010) (supplementary material Fig. S1D-G). The Otx2–/–;R26GFP/+ ESC line carries an Otx2floxed-out null allele and the Otx2lacZ null allele; in addition, the GFP gene was inserted into the Rosa26 locus for chimerism studies (supplementary material Fig. S1H-O). The R26GFP/+ ESC line carries the GFP gene in the Rosa26 locus and was used as control for chimerism experiments (supplementary material Fig. S1M-O). The Otx2flox/–;R26CreER/+ ESC line carries an Otx2flox allele in combination with the Otx2lacZ null allele (Otx2flox/–). An inducible CreER-puro cassette was introduced into the Rosa26 locus (supplementary material Fig. S1P-S).

ESC differentiation

Serum-free floating embryoid body-like aggregates (SFEBs) were generated from ESCs as reported (Watanabe et al., 2005). Mesendoderm differentiation in adherent conditions was obtained under serum-free conditions in the presence of activin A for 4 days (d) (Waese and Stanford, 2011; Izumi et al., 2007). For neural differentiation, SFEBs were cultured up to d5 without exogenous factors or with Dkk1 (from d1 to d5) (R&D Systems; 500 ng/ml), followed by adherent culture for 5 more days (Watanabe et al., 2005); alternatively, neural differentiation was induced using adherent monoculture conditions (Ying et al., 2003b).

ESC transfection

Otx2–/– ESCs were transfected using Lipofectamine 2000 reagent (Invitrogen) with a plasmid overexpressing Otx2 cDNA under a CMV-chicken β-actin promoter (CAG). Six hours after transfection, ESCs were seeded in ESC medium or differentiated into SFEBs.

ESC-derived and embryo-derived EpiSCs

ESCs were seeded at a density of 3×103/cm2 in N2B27 supplemented with 20 ng/ml activin A (R&D Systems) and 12 ng/ml Fgf2 (Peprotec) and cultured for 6 days (Brons et al., 2007; Tesar et al., 2007; Zhang et al., 2010), which corresponded to passage (p) 0. For further passages, EpiSCs were cultured in the same conditions. For Otx2 conditional inactivation, Otx2flox/–;R26CreER/+ EpiSCs were administered with 4-hydroxy-tamoxifen (4-OHT) (125 nM) at p1 and kept in 40 nM 4-OHT throughout subsequent passages. Epiblasts were dissected from single embryonic day (E) 5.75 mouse embryos and incubated in EpiSC medium.

Growth factors and cytokine assays

LIF and FGF response was monitored as previously described (Mitsui et al., 2003; Zhang et al., 2010). For experiments involving BMP4 (recombinant human; R&D Systems), the factor was administered on d1 to ESC- or EpiSC-derived SFEBs (Zhang et al., 2010) at 0.5 and 10 ng/ml; SFEBs were then cultured up to d5. EpiSCs induced with Fgf2 and activin A were also administered with BMP4 (10 ng/ml) at p1 and until p4. Experiments involving activation or inhibition of the FGF and BMP pathways were performed in three conditions: (1) ESCs kept in 15% knockout serum replacement (KSR) medium with LIF were administered with BMP4 (10 ng/ml) or Fgf2 (12 ng/ml) or the BMP inhibitor dorsomorphin (DM) (0.5 μM; Calbiochem) or the Fgf/Erk inhibitor PD325901 (PD) (1 μM; Calbiochem) or with both inhibitors for 12 hours; (2) ESCs kept in ESC medium were cultured for the last 12 hours in 5% KSR medium plus BMP4 or Fgf2 or their inhibitors; (3) EpiSCs in N2B27 containing Fgf2 and activin A were cultured for 12 hours in N2B27 plus activin A only or also supplemented with BMP4 or DM, PD, or DM plus PD.

Alkaline phosphatase (ALP) assays and LIF dependence experiments

ALP staining was performed on EpiSCs at p0 and later passages or on ESCs in three different growth conditions at clonal density (1×103 cells/10 cm2): in ESC medium (with LIF), without LIF, or without LIF plus JAK inhibitor 1 (0.6 μM; Calbiochem). ALP activity was revealed by the naphthol/Fast Red Violet reaction.

EpiSCs derived from SFEBs

SFEBs treated with BMP4 (10 ng/ml) from d1 were trypsinized at d2 and reseeded in EpiSC medium (without BMP4) at a cell density of 1×105/10 cm2. Immunostaining for Oct4 was performed on d6 to unambiguously identify EpiSC colonies.

Chimeras and teratomas

Chimeras were obtained by injecting 13-16 ESCs or Otx2–/– EpiSCs into C57BL/6 blastocysts and colonization was evaluated by assessing GFP expression on E11.5, E9 or E8. Teratomas were generated by subcutaneous injection of 1.5×106 ESCs or EpiSCs into NOD SCID mice. For Otx2–/– ESCs, teratoma occurrence was also assayed by injecting 6×106 ESCs.

Cell counting and statistical analysis

Cell counting was performed manually on immunohistochemistry images printed in A4 format or using ImageJ software (NIH). Standard deviation was calculated from four independent experiments.

RESULTS

Otx2 is expressed in preimplantation and early postimplantation embryos, ESCs and EpiSCs

In preimplantation mouse embryos, Otx2 was expressed in late morula blastomeres co-expressing Nanog or the trophectoderm determinant Cdx2 and, during blastocyst development, Otx2 colocalized with Cdx2 in trophectoderm and with Nanog in a fraction of cells of the ICM (Fig. 1A). Upon embryo implantation, robust Otx2 expression was detected in epiblast and visceral endoderm. At E6.5, Otx2 was downregulated in posterior epiblast, where the second wave of Nanog expression was activated (Fig. 1B). In ESCs, 46% of the Oct4+ cells co-expressed Otx2, whereas only a fraction of Oct4+ cells colocalized with Nanog and Klf4 (Fig. 2A-C). Cell counting on cytospun ESCs showed that Otx2+ cells were almost equally distributed between those expressing high or moderate levels of Nanog and those with low or no expression of Nanog (Fig. 2D-G; supplementary material Table S2). Moreover, ESCs exhibiting high levels of Otx2 preferentially expressed low levels of Nanog, whereas those with low levels of Otx2 exhibited high Nanog expression (Fig. 2D-G). In EpiSCs, Otx2 was detected in all Oct4+ cells at a level that varied between cells and was frequently complementary to that of Nanog (Fig. 2H). These data indicate that Otx2 is a molecular correlate of different pluripotent cell types in vivo and in vitro.

Otx2 expression in preimplantation and early postimplantation mouse embryos. (A) Co-immunohistochemistry for Otx2 and Nanog and for Cdx2 and Otx2 in late morula embryos and during blastocyst development. (B) Co-immunohistochemistry for Otx2 and Nanog in early postimplantation embryos up to early streak stage. The arrows in A point to Nanog+ Otx2+ ICM cells and Otx2+ Cdx2+ TE cells. ICM, inner cell mass; TE, trophectoderm; Epi, epiblast; VE, visceral endoderm.

Otx2 expression in ESCs and EpiSCs. (A-C) Immunohistochemistry showing that in ESCs Otx2 is co-expressed with Oct4 (A) and only with a fraction of cells expressing Nanog and/or Klf4 (B,C). (D-G) Cell counting of cytospun ESCs immunostained for Otx2 and Nanog (D-F) shows the percentage of total Otx2+ cells (regardless of the Otx2 expression level) co-expressing high or low levels of Nanog (G); the cell counting also shows that ESCs with high levels of Otx2 preferentially co-express low levels of Nanog, whereas those exhibiting low levels of Otx2 co-express high levels of Nanog (G). Error bars indicate s.d. (H) Immunohistochemistry for Otx2 and Oct4 and for Otx2 and Nanog show that Otx2 is an EpiSC marker.

Otx2 is an early responding factor to ESC differentiation

ESC differentiation may be activated by LIF withdrawal and/or low concentrations of serum, and early postimplantation embryonic development can be mimicked by ESC-derived embryoid bodies. In these culture conditions, Otx2 expression was highly responsive to ESC differentiation. Indeed, LIF withdrawal and/or diminished concentrations of KSR medium induced a rapid and generalized activation of Otx2 and a corresponding reduction in the number of Nanog+ ESCs (supplementary material Fig. S2A-D). Similarly, in differentiating SFEBs, Otx2 rapidly spread to all the Oct4+ cells, mirrored the downregulation of Nanog, Klf4 and Rex1 (Zfp42 – Mouse Genome Informatics), and anticipated the induction of the epiblast markers Fgf5 and Cer-l (Cer1 – Mouse Genome Informatics) and the generation of T+ primitive streak-like and Sox1+ neural cells (supplementary material Fig. S2E-H, Table S3). These observations support the possibility that Otx2 is required in ESCs to control their state and/or promote their differentiation.

Otx2 is required to maintain the ESC state

To investigate the role of Otx2 we generated mutant ESC lines that lack (Otx2–/–) or ubiquitously and constitutively express (R26Otx2/Otx2) Otx2 (supplementary material Fig. S1). First, we studied functional parameters and markers related to the undifferentiated state or predisposition to differentiation. Compared with wild type (wt), virtually all the Otx2–/– ESC colonies exhibited a sphere-like morphology, uniform ALP staining, ubiquitous distribution of Nanog and Klf4 and higher expression of Rex1 (Fig. 3A-D; supplementary material Tables S4, S5). During the earliest stages of wt ESC colony formation, Otx2 and Nanog exhibited complex expression profiles with variable degrees of complementarity, whereas Otx2–/– ESC colonies exhibited constitutively high expression of Nanog (supplementary material Fig. S3). These data suggest that, in the absence of Otx2, the fluctuating expression of Nanog is severely affected and, therefore, that Otx2 is directly or indirectly required to prevent its constitutive expression.

Maintenance of the ESC state depends on Otx2. (A-D) Compared with wt, virtually all of the Otx2–/– ESC colonies show uniform ALP staining (A,B), ubiquitous high expression of Nanog and Klf4 (A,C,D) and increased levels of Rex1 transcripts (D); conversely, R26Otx2/Otx2 ESCs show decreased numbers of colonies with uniform ALP staining (A,B), a diminished percentage of total cells expressing Oct4, Nanog or Klf4 (A,C), low expression of Rex1 (D) and activation of the epiblast markers Fgf5, Cer-l and T (A,D). Arrowheads (A) point to ESC colonies that are not uniformly ALP+. (E) Compared with wt, Otx2–/– and R26Otx2/Otx2 ESCs respectively exhibit higher and lower endogenous LIF activity as revealed by p-Stat3 level; when stimulated with LIF after 60 minutes of LIF deprivation, the p-Stat3 level is substantially increased in Otx2–/– and attenuated in R26Otx2/Otx2 ESCs. (F) Compared with wt ESCs, the endogenous FGF activity revealed by p-Erk1,2 is diminished in Otx2–/– and substantially increased in R26Otx2/Otx2 ESCs. (G,H) LIF deprivation, or LIF deprivation in the presence of JAK inhibitor 1, is not sufficient to abolish self-renewal (G) and ESC colonies continue to exhibit uniform ALP staining (H). β-actin was used to normalize western blots (E,F, Nanog and T in D) and RT-PCRs (Klf4, Rex1, Fgf5 and Cer-l in D). Error bars indicate s.d. (I) The ESC state is characterized by dynamic and reversible fluctuations between ESCs with high self-renewal and those with reduced self-renewal that are poised to become postimplantation epiblast. Antagonism driving the metastable ESC state is primarily controlled by FGF and LIF signaling activities together with Nanog. Our results suggest that Otx2 is a novel component of this circuit, being required to maintain the metastable ESC condition by opposing high self-renewal and promoting predisposition to differentiation.

In Otx2–/– ESCs, the endogenous activities of LIF and FGF were respectively enhanced and severely decreased, as monitored by the level of the phosphorylated, active forms of the LIF signaling transcriptional mediator Stat3 (p-Stat3) and the extracellular signal-related kinases 1 and 2 (p-Erk1,2) (Fig. 3E,F). Furthermore, compared with wt, LIF addition to LIF-deprived ESCs generated a substantially higher response in Otx2–/– ESCs (Fig. 3E), and LIF deprivation and blockade of Stat3 phosphorylation by JAK inhibitor 1 were not sufficient to disrupt the undifferentiated state of Otx2–/– ESCs (Fig. 3G,H; supplementary material Table S4). Since this phenotype may also depend on loss of autocrine Fgf4 activity (Kunath et al., 2007) or abnormal expression of NuRD complex components (Reynolds et al., 2012; Kaji et al., 2006), the expression of Fgf4, Fgfr1-4, Mbd3, Mta1 and Mta2, Rbap46 (Rbbp7 – Mouse Genome Informatics) and Hdac1 was analyzed, and showed a very mild reduction in Otx2–/– ESCs only for Fgf4 (supplementary material Fig. S4).

Chimerism and teratoma formation. (A-C) GFP fluorescence and immunohistochemistry show that the efficiency in generating chimeric embryos is very similar for R26GFP/+ and Otx2–/–;R26GFP/+ ESCs, and is substantially affected for R26Otx2/Otx2 ESCs, even when scoring E8 embryos (A,C). Note that highly chimeric embryos injected with Otx2–/–;R26GFP/+ cells exhibit a typical headless phenotype (B), whereas in those that are less chimeric, Otx2–/– ESCs fail to colonize forebrain and midbrain (C). Arrows (C) point to isolated patches of R26Otx2/Otx2 ESCs. (D,E) The frequency of teratoma occurrence (D) and their growth (E) are severely affected in Otx2–/– ESCs, whereas these parameters are similar for R26Otx2/Otx2 and control ESCs. fb, forebrain; mb, midbrain; hb, hindbrain; sc, spinal cord; he, heart.

Next, we investigated whether ubiquitous and constitutive expression of Otx2 was sufficient to affect the ESC state by promoting differentiation. R26Otx2/Otx2 ESCs exhibited a fairly flat morphology, a low frequency of uniformly ALP-stained colonies, a reduced percentage of ESCs co-expressing high levels of Nanog and Klf4, decreased expression of Rex1 (Fig. 3A-D), a heavily attenuated response to LIF and substantially enhanced activity of endogenous FGF (Fig. 3E,F). Remarkably, R26Otx2/Otx2 ESCs exhibited the typical signature of primed EpiSCs, such as the expression of Fgf5 and Cer-l, generation of T+ cells (Fig. 3A,D) and very poor ability to generate chimeras, while retaining high efficiency in teratoma formation (Fig. 4A,C-E). Thus, Otx2 ubiquitous expression in ESCs is sufficient to induce stable molecular and functional features of the EpiSC state.

Collectively, these findings suggest that Otx2 is required to maintain the integrity of the ESC state by controlling the dynamic balance between pluripotency with high self-renewal and susceptibility to differentiation (Fig. 3I). In this context, Otx2–/– ESCs resemble ESCs cultured in 2i, which exhibit severe downregulation of Otx2 (Marks et al., 2012).

Otx2 is required for ESC conversion into EpiSCs

We then studied Otx2 requirement in ESC differentiation by monitoring sequential steps marking pluripotency transition from ESCs to EpiSCs. First, we analyzed the response to Fgf2, which primes ESC differentiation. Compared with wt, Otx2–/– ESCs howed a severe reduction in the p-Erk1,2 level, whereas in R26Otx2/Otx2 ESCs, which already exhibited strong endogenous FGF activity, the response to Fgf2 was substantially higher (Fig. 5A). In the absence of any growth factor or cytokine, Otx2–/– differentiating SFEBs showed after d1 a rapid loss of Oct4+ cells, failed to efficiently downregulate Nanog, Klf4 and Rex1, did not activate Fgf5 and Cer-l, and prematurely differentiated exclusively into Sox1+ neural cells co-expressing nestin and Sox2 (Fig. 5B-D; supplementary material Fig. S5A, Tables S3, S8). Conversely, R26Otx2/Otx2 SFEBs showed a more graded decrease of Oct4+ cells, efficiently repressed Nanog, Klf4 and Rex1, expressed high levels of Fgf5 and Cer-l over the entire timecourse and differentiated prevalently into T+ primitive streak-like cells (Fig. 5B-D; supplementary material Fig. S5A, Tables S3, S8). Compared with d3.5 control SFEBs, these R26Otx2/Otx2 SFEBs exhibited a substantial increase in the number of Sox1– Foxa2+ cells (Fig. 5D), which are likely to correspond to T+ cells generated before d3.5 and differentiated into endoderm. Further mesendoderm determinants, such as Mixl1, Eomes, Gsc and Tbx6 (Izumi et al., 2007), showed that, as for T, their expression was virtually lost in Otx2–/– SFEBs (supplementary material Fig. S5B). Thus, in the absence of any added factor, Otx2 is required in ESCs to promote their exit from the undifferentiated state and to protect committed EpiSCs from a premature neural fate.

Otx2 is required for ESC differentiation. (A) Compared with wt ESCs stimulated with Fgf2 after a brief starvation in 5% KSR, the response of Otx2–/– ESCs to Fgf2 is substantially affected, as revealed by the p-Erk1,2 level, and is even more pronounced for R26Otx2/Otx2 ESCs, which already exhibit an elevated level of endogenous p-Erk1,2. (B) Oct4+ cell counts over the entire timecourse. (C) Expression analysis in wt, Otx2–/– and R26Otx2/Otx2 differentiating SFEBs for Nanog, Klf4, Rex1, Fgf5, Cer-l, T and Sox1. β-actin is employed to normalize western blots and RT-PCRs (Klf4, Rex1, Fgf5 and Cer-l in C). (D) Sox1+, T+, and Sox1– Foxa2+ SFEB cell counts at d3.5. Error bars indicate s.d.

The phenotype of Otx2–/– ESCs is not caused by a secondary adaptive effect

To investigate whether the abnormalities described for Otx2–/– ESCs were caused by a secondary adaptive effect triggered by Otx2 ablation, we first analyzed whether reintroduction of Otx2 is sufficient to rescue some of the major phenotypic abnormalities and, second, inactivated Otx2 in a 4-OHT conditionally inducible ESC line (Otx2flox/–;R26CreER/+) (supplementary material Fig. S1). Immunostaining of Otx2 and Nanog in Otx2–/– ESCs transfected with a pCAG-Otx2 plasmid showed that most of the cells expressing high levels of Otx2 lacked or exhibited severely reduced expression of Nanog; moreover, d3.5 SFEBs generated after the pCAG-Otx2 transfection partially recovered the differentiation of T+ and Eomes+ cells (supplementary material Fig. S7A,B). In the second experiment, 48 hours after 4-OHT administration the Otx2flox/–;R26CreER/+ ESC colonies showed a compact morphology, ubiquitous expression of Nanog, increased levels of endogenous p-Stat3 and a moderate decrease in p-Erk1,2; SFEBs derived from 4-OHT-treated ESCs almost completely lacked T+ and Eomes+ cells (supplementary material Fig. S7C-E). Thus, the major phenotypes of Otx2–/– ESCs are not due to adaptive and irreversible effects triggered by Otx2 inactivation.

Otx2 stabilizes the EpiSC state

To investigate which, if any, of the Otx2 requirements described in differentiating SFEBs might be detected in vivo, we revisited the phenotype of Otx2–/– embryos. Otx2–/– embryos showed widespread derepression of Nanog in the epiblast of pre-gastrula and gastrulating embryos (Fig. 6A-C), while the generation of T+ cells, although delayed, was still efficient and premature differentiation of Sox1 neural progenitors was not observed (Fig. 6; data not shown) (Acampora et al., 2009; Acampora et al., 1995; Ang et al., 1996). We hypothesized that in vivo, with the exception of Nanog, embryonic extrinsic signals might circumvent abnormalities detected in Otx2–/– differentiating SFEBs. We examined whether Otx2–/– ESCs may be converted into EpiSCs when provided with Fgf2 and activin A, which promote ESC transition into EpiSCs and maintain their undifferentiated state. Moreover, FGF activity is required in EpiSCs also to suppress neuroectoderm differentiation and prevent regression to a preimplantation ESC-like state (Lanner and Rossant, 2010; Greber et al., 2010; Greber et al., 2011; Kunath et al., 2007).

Otx2–/– embryos exhibit Nanog but not Sox1 derepression. (A) Immunohistochemistry for Otx2 and Nanog on wt and Otx2–/– embryos shows that in early postimplantation Otx2–/– embryos the expression of Nanog is activated in most epiblast cells. (B,C) Immunohistochemistry for Oct4 and Nanog, T and Nanog, and T and Foxa2 on wt and Otx2–/– embryos at E6.5 (B) and E7.5 (C).

Otx2–/– EpiSC colonies at p0, which corresponds to d6 of differentiation, showed spotted ALP staining and a mild increase in Sox1 expression but fairly normal expression of Nanog, Fgf5, Cer-l and T (supplementary material Fig. S8A-C). Suspecting these abnormalities as initial signs of a much more serious instability, Otx2–/– EpiSCs were analyzed at subsequent passages. Strikingly, at p5, Otx2–/– EpiSCs showed intense ALP staining and high levels of Nanog, mild reduction of Fgf5 and Cer-l transcripts in Oct4 normalized RNA samples, increased generation of Sox1+ nestin+ cells and a corresponding decrease of T+ cells (Fig. 7A,B). This phenotype worsened severely over time and, at p10, the great majority of Otx2–/– EpiSC colonies showed domed or sphere-like morphology with uniform ALP staining, strong Nanog immunoreactivity, severe decrease in Fgf5 and Cer-l expression, almost complete extinction of T+ cells, substantial reduction in the expression of Eomes, Mixl1, Gsc and Tbx6, and massive derepression of the neural lineage (Fig. 7A,B). However, p12 Otx2–/– EpiSCs can be propagated and maintained in EpiSC medium, thus excluding the possibility that they might have regressed to LIF dependence (data not shown). Accordingly, p5 Otx2–/–;R26GFP/+ EpiSCs failed to generate chimerism in E9 embryos (n=24; data not shown). However, p0 and p5 Otx2–/– EpiSCs were able to form teratomas and, as for those derived from Otx2–/– ESCs, they generated neuronal, glial, muscle-like and endodermal derivatives and numerous Sox1+ nestin+ rosette-like neural progenitors and Oct4+ Nanog+ cell aggregates (supplementary material Fig. S9, Table S7).

Otx2 is required for maintenance of the EpiSC state. (A) Expression analysis of Klf4, Rex1, Fgf5, Cer-l, Sox1 and T markers in wt and Otx2–/– ESCs, d2 SFEBs, and EpiSCs at passage (p) 5 and p10, and expression of Eomes, Mixl1, Gsc and Tbx6 mesendoderm markers in wt and Otx2–/– EpiSCs. (B) ALP reactivity and immunostaining on wt EpiSCs at p10 and Otx2–/– EpiSCs at p5 and p10 for Nanog and Oct4, Sox1 and Oct4, T and Oct4, Sox1 and nestin, and Sox1 and Tuj1. (C) ALP staining and immunohistochemistry for Nanog and Oct4, Sox1 and Oct4, and T and Oct4 on Otx2flox/–;R26CreER/+ EpiSCs untreated or treated at p1 with 4-OHT and analyzed at p4 and p8. (D,E) RT-PCR and western blot assays showing full inactivation of Otx2 at p2 (D) and expression of Fgf5, Cer-l, Sox1 and T at p4 and p8 (E). (F) Otx2–/– embryo-derived EpiSCs show at p6 a phenotype that is apparently identical to that of ESC-derived EpiSCs. RT-PCRs are normalized by Oct4 and western blots by β-actin.

Next, we analyzed the effect of Otx2 ablation or constitutive expression following a different protocol using only activin A for mesendoderm differentiation (Waese and Stanford, 2011; Izumi et al., 2007), and found that lack of Otx2 primarily affected the expression of Fgf5, Eomes, Mixl1, Gsc and Foxa2 and, interestingly, induced Sox1 and nestin in numerous Oct4+ patches; conversely, mesendoderm markers were all upregulated in R26Otx2/Otx2 cells (supplementary material Fig. S10). Together, these data indicate that a high dosage of Fgf2 and activin A apparently compensates for the Otx2 requirement only for the initial transition of ESCs to primed EpiSCs, but not for maintenance of EpiSC identity. To assess whether Otx2 is an intrinsic stabilizer of the EpiSC state, we analyzed at p4 and p8 the phenotype of Otx2flox/–;R26CreER/+ conditional EpiSCs administered at p1 with 4-OHT, and found that they exhibited the same abnormalities described for Otx2–/– EpiSCs at similar passages (Fig. 7C-E). Of note, Otx2flox/–;R26CreER/+ EpiSC colonies that were not treated with 4-OHT also showed mild impairments at p8, suggesting an Otx2 dosage requirement for maintenance of the EpiSC state (Fig. 7C-E).

Finally, we tested our hypothesis that, in vivo, extrinsic factors might circumvent the requirement for Otx2 in EpiSCs. EpiSCs isolated from the egg cylinder of Otx2–/– embryos developed severe abnormalities that were undistinguishable from those of Otx2–/– ESC-derived EpiSCs (Fig. 7F). These findings indicate that Otx2 is an intrinsic determinant required to stabilize the EpiSC state by repressing the progressive switch of mesendoderm to neural fate and suggest that factor(s) other than, or together with, Fgf2 and activin A are required to stabilize the EpiSC state in cooperation with Otx2.

Otx2 functional interactions with Fgf2 and BMP4 for ESC transition to EpiSC and maintenance of the EpiSC state through suppression of neural fate

Antagonism and synergism between BMP4 and Otx2 and cooperative recovery by BMP4 and Fgf2 of Otx2 requirement in EpiSCs. (A-C) Sox1– Oct4+ (A), Sox1+ Oct4– (B) and Sox1– Oct4– (C) cell counts in wt, Otx2–/– and R26Otx2/Otx2 ESC-derived and EpiSC-derived d5 SFEBs untreated (no factor) or administered with the indicated dosage of BMP4. (D) Cell counting of EpiSC colonies derived from d2 BMP4-treated wt and Otx2–/– SFEBs. (E) Immunostaining for Oct4 and Sox1, and Oct4 and T, performed on Otx2–/– EpiSCs cultured in the presence of Fgf2 and activin A and receiving, from p1 to p4, also BMP4 (10 ng/ml). (F) Western blots showing that Otx2–/– EpiSCs receiving BMP4 at p1 exhibit at p4 only mild expression of Sox1 and a normal level of T. Western blots are normalized by β-actin. Error bars indicate s.d.

To assess the hierarchical position of Otx2 in this process, we analyzed its expression in response to BMP4 or Fgf2 and their inhibitors (DM and PD, respectively) in ESCs cultured in 15% KSR with LIF and in ESCs primed to differentiate in 5% KSR. The Otx2 response was also compared with that of Nanog. Fgf2 induced robust activation of Otx2 in ESCs cultured in 15% KSR with LIF and even stronger activation in those kept in 5% KSR; accordingly, treatment with the Fgf inhibitor PD generated the opposite effect, and treatment with BMP4 or its inhibitor DM revealed a moderate repressive effect of BMP4 on Otx2 expression (supplementary material Fig. S12A-C). The effects on Nanog expression mirrored those on Otx2. Together, these findings suggest that, during the initial ESC transition to EpiSCs, Otx2 antagonizes the inhibitory action of BMP4 through a positive loop with Fgf2 (supplementary material Fig. S11E).

Then, we tested whether BMP4 requires Otx2 to suppress neural fate in SFEBs, this time derived from p0 EpiSC colonies. Compared with wt, the BMP4 antineuralizing activity was less efficient in Otx2–/– EpiSC-derived SFEBs (Fig. 8A-C; supplementary material Fig. S11C). We reasoned that if the inefficient neural suppression by BMP4 reflected lack of cooperation with Otx2, then the BMP4 anti-neuralizing activity should be potentiated in R26Otx2/Otx2 ESC-derived SFEBs. Indeed, R26Otx2/Otx2 SFEBs cultured with a very low concentration of BMP4 (0.5 ng/ml) that is unable to suppress neural fate in wt SFEBs, fully suppressed Sox1+ neural cells and generated almost exclusively Oct4– Sox1– cells (Fig. 8A-C; supplementary material Fig. S11D). Accordingly, when Otx2–/– EpiSCs induced by Fgf2 and activin A also received BMP4 at p1, the derepression of neural fate was inhibited and EpiSC identity was maintained (Fig. 8E,F). This also suggests that, in vivo, the same compensatory mechanism might stabilize the identity of the epiblast of Otx2–/– embryos up to gastrulation.

To gain more insight into this aspect, we investigated Otx2 expression in EpiSCs in response to Fgf2, BMP4 and their inhibitors. The expression of Otx2 detected in the presence of Fgf2 and activin A was unaffected by Fgf2 withdrawal or Fgf2 withdrawal and addition of BMP4 (supplementary material Fig. S12D,E). However, treatment with DM in the presence of activin A induced a moderate activation of Otx2, which was enhanced by PD or DM plus PD; of note, Otx2 activation correlated with downregulation of Nanog and Oct4 (supplementary material Fig. S12D,E). Thus, in contrast to ESCs, inactivation of FGF and/or BMP4 signaling in EpiSCs upregulates Otx2 and downregulates pluripotency factors. These data are similar to those reported in human ESCs showing that inhibition of FGF signaling is reflected in OTX2 activation, repression of NANOG and OCT4 and OTX2-mediated activation of PAX6 (Greber et al., 2011). Collectively, these experiments suggest that, in EpiSCs, Otx2 expression is maintained at a relatively low level at which it synergizes with Fgf2 and BMP4 to stabilize the EpiSC state by suppressing neural fate (supplementary material Fig. S11E).

Otx2 requirement for ESC differentiation into anterior neuroectoderm

Previous studies indicated the crucial role played by Otx2 in the specification, regionalization and differentiation of the rostral neuroectoderm (Simeone et al., 2002; Acampora et al., 2009; Simeone et al., 2011). We examined whether the identity of neural progenitors is affected by Otx2. First, we induced the differentiation of neural progenitors enriched in telencephalic precursors (Watanabe et al., 2005). Compared with wt, in Otx2–/– neural cells, the expression level of the pan-telencephalic marker Foxg1 was severely diminished, even when telencephalic differentiation should be enhanced by treatment with the WNT antagonist Dkk1; conversely, in R26Otx2/Otx2 neural progenitors the expression of Foxg1 was moderately increased (Fig. 9A). Otx2–/– neural cells retained expression of En1 and En2, and essentially lacked that of telencephalic, diencephalic and mesencephalic markers; conversely, R26Otx2/Otx2 neural cells showed increased expression of anterior neuroectoderm markers and attenuated expression of En1 and En2 (Fig. 9A).

Otx2 is required for specification of anterior neuroectoderm. (A) Wt, Otx2–/– and R26Otx2/Otx2 ESCs induced toward neural differentiation without or with Dkk1 and assayed for the expression of the pan-telencephalic (Tel) marker Foxg1, the pallial markers Emx1, Tbr1 and Emx2, the diencephalic (Di) and mesencephalic (Mes) marker Dmbx1, and the posterior mesencephalic and anterior metencephalic (Met) markers En1 and En2. (B,C) Immunohistochemistry for Foxg1 and Pax6 and for Foxg1 and Nkx2.1 (B) and cell counting analysis (C) to determine the pallial (Pax6+) and subpallial (Nkx2.1+) identity of Foxg1+ cells in wt, Otx2–/– and R26Otx2/Otx2 ESC-derived neural precursors. Error bars indicate s.d. (D) Neural differention in adherent monolayer culture conditions shows that, compared with wt, the expression of telencephalic, diencephalic and mesencephalic markers is reduced in Otx2–/– and increased in R26Otx2/Otx2 neural cells, the expression of posterior mesencephalic and rostral metencephalic markers is not substantially affected in Otx2 mutant cells, and that of metencephalic and spinal cord (SC) markers is increased in Otx2–/– and diminished in R26Otx2/Otx2 neural cells. RT-PCRs are normalized by β-actin.

A different protocol of ESC differentiation into neuroectoderm precursors in adherent monoculture (Ying et al., 2003b) confirmed that lack of Otx2 results in decreased expression of telencephalic, diencephalic and mesencephalic markers and revealed increased expression of metencephalic and spinal cord markers; the opposite expression profile was observed in R26Otx2/Otx2 ESC-derived neural cells (Fig. 9D). Thus, these findings indicate that, as in the embryo, Otx2 is crucially required to confer anterior character to ESC-derived neuroectoderm progenitors.

In this study, we have investigated for the first time whether Otx2, which has been extensively investigated in the context of brain development, is required in ESCs and EpiSCs. Otx2 is expressed in a large subset of ESCs that defines a cell population showing graded transition from those expressing high levels of Nanog to those exhibiting low or no Nanog expression. ESC culture conditions promoting differentiation (LIF withdrawal and/or Fgf2 addition and/or BMP4 inhibition) induce rapid activation of Otx2, which is prevented by FGF inhibition. Based on these observations, we hypothesized that genetic ablation of Otx2, or its constitutive and ubiquitous expression, might affect the ESC state and corrupt the metastable condition. Lack of Otx2 indeed causes severe abnormalities consisting of ubiquitous and constitutive high level expression of Nanog and Klf4, increased LIF signaling and weakened activity of FGF signaling; conversely, constitutive and ubiquitous activation of Otx2 causes a substantial reduction in the number of ESCs co-expressing high levels of Nanog and Klf4, a robust increase of FGF activity, induction of epiblast markers and a very poor ability to generate chimeric embryos. These data strongly suggest that Otx2 is a novel intrinsic determinant of the ESC state, controlling the balance between self-renewal and differentiation. We propose that Otx2 protects the susceptibility to differentiation by counter-balancing signaling pathways and transcription factors that promote ground state pluripotency. Our findings suggest that Otx2 and Nanog, by promoting antagonistic conditions, together contribute to define the ESC metastable state (Fig. 3I). Of note, lack of Otx2, or its ubiquitous activation, generate ESC abnormalities that are similar to those exhibited by ESCs overexpressing or lacking Nanog, respectively (Silva and Smith, 2008; Chambers et al., 2007; Chambers et al., 2003; Mitsui et al., 2003).

This study shows that Otx2 is also required for ESC transition into EpiSCs and maintenance of the EpiSC state. In the absence of any added factor, Otx2–/– ESCs fail the transition to EpiSCs and prematurely differentiate only into neural cells. When provided with Fgf2 and activin A, transition of Otx2–/– ESCs to EpiSCs is initially achieved but maintenance of their state in terms of identity and fate is gradually lost through a progressive mesendoderm-to-neural fate switch accompanied by loss of EpiSC markers, strong activation of Nanog and intense ALP reactivity. Similar abnormalities, with early derepression of neural fate and selective impairment of the endoderm lineage, were also observed in Otx2–/– ESCs induced only with activin A. Thus, Otx2 represents a key factor that is intrinsically required to maintain EpiSC identity, primarily by suppressing neural fate and preventing the adoption of features resembling naïve pluripotency. We speculate that Otx2–/– EpiSCs might progressively regress to an early postimplantation-like epiblast stage, similar to that at which SFEBs prematurely derepress neural fate. In the absence of Otx2, maintenance of EpiSC identity and suppression of neural fate require a high dosage of both Fgf2 and BMP4 in the presence of activin A. Like Fgf2, BMP4 also plays a dual role, first opposing ESC transition into EpiSCs and then suppressing neural fate in EpiSCs (Zhang et al., 2010). Our data suggest that, during the first stage Otx2 antagonizes BMP4-mediated inhibition of EpiSC derivation and, subsequently, in EpiSCs Otx2 synergizes with BMP4 for suppression of neural fate (supplementary material Fig. S11E).

Otx2 has been extensively studied in the context of its essential role in the development and differentiation of anterior neuroectoderm. Our data indicate that Otx2 is crucially required in ESC-derived neural cells to specify the identity of telencephalic and mesencephalic territories and to allow chimerism in the neuroectoderm rostral to the isthmic organizer.

In summary, this study has revealed that Otx2 is a novel determinant controlling the ESC state and its transition to a stable EpiSC condition. Subsequently, as in vivo, Otx2 is required to specify the anterior identity of ESC-derived neural progenitors.